Synthesis of cyclic amidines

ABSTRACT

The invention relates to an innovative method for synthesis of cyclic amidines. The synthesis starts from a β-, γ- or δ-lactone which is twofold brominated. After esterification of the carboxyl function, the bromine atoms are nucleophilically substituted and the corresponding diamino compound is obtained. The ring closure to the cyclic amidine is accomplished subsequently by reaction with orthoester, imidate or thioimidate. Owing to interposing additional steps for recovery of the diamino compound in enantiomerically pure form, the enantiomers of the cyclic amidines can be stereoselectively synthesized.

The invention relates to a process for the preparation of cyclicamidines. The invention furthermore relates to various cyclic amidinesthemselves. In particular, the cyclic amidines concerned may be ectoine,ectoine derivates, and ectoine analogues.

Osmolytes and/or compatible solutes from extremophilic microorganismsconstitute a well known group of low-molecular immunising substances.Extremophiles are very extraordinary microorganisms because they growoptimally and/or with high salt concentrations (up to 200 g NaCl/I) andhigh temperatures (60 to 110° C.), which in case of mesophilic (normal)organisms would lead to massive damage to cellular structures. In recentyears, substantial research efforts have therefore been pursued toidentify the biochemical components that lead to a remarkablestabilization of cell structures. Though a great deal of enzymes fromhyperthermophilic microorganisms remain stable even under hightemperatures, this cannot be generalized for cellular structures ofthermophilic and hyperthermophilic organisms. Low-molecular organicsubstances (compatible solutes, osmolytes) in the intra-cellularenvironment render a substantial contribution to the high temperaturestability of cell structures. Various novel osmolytes could beidentified in recent years for the first time ever in extremophilicmicroorganisms. In some cases, it has already been managed todemonstrate the contribution rendered by these compounds to theprotection of cellular structures—above all enzymes—against heat anddryness (K. Lippert, E. A. Galinski, Appl. Microbiol. Biotech. 1994, 37,61-65; P. Louis, H. G. Trüper, E. A. Galinski, Appl. Microbiol. Biotech.1994, 41, 684-688; Ramos et al., Appl. Environm. Microbiol. 1997, 63,4020-4025; Da Costa, Santos, Galinski, Adv. in Biochemical EngineeringBiotechnology, 61, 117-153).

For a lot of compatible solutes, sensible applications have come up andrealized in the medical, cosmetic, and biological field. Reckoned to beamong the most important compatible solutes is ectoine(1,4,5,6-tetrahydro-2-methyl-pyrimidine-4-carboxylic acid) and/or itsderivatives. For example, EP 0 887 418 A2 describes the use of ectoineand hydroxyectoine to treat skin diseases or as an effective additivefor cryoprotection of biological active ingredients and cells. DE 102006 056 766 A1 outlines the use of ectoines to treat the vascular leaksyndrom (VLS). Further examples are the stabilization of vaccines (DE100 65 986 A1), the treatment of pulmonary diseases due to the impact ofsuspended particulate matter and cardiovascular diseases (DE 103 30 768A1) or the dermatological use to treat neurodermatitis (DE 103 30 243A1).

Under high salt concentration conditions, compatible solutes likeectoines and hydroxyectoines can be enriched very well in bacteria likehalomonas elongata or marinococcus halophilus and subsequently isolatedfrom the dry mass. One possibility is the so-called “bacteria milking”method in which the salinity of a medium is decreased after the cellswith high salinity have produced large quantities of ectoine. Decreasingthe salinity causes the bacteria to sluice-out ectoine into the mediumfrom which it can be isolated and chromatographically purified. Thecells themselves remain intact and can be “milked” several times (T.Sauer, E. A. Galinski, Biotech. Bioeng., 1998, 57, 306-313). Theadvantage of such a method as compared with a purely chemical synthesisin particular is the stereoselectivity of the biosynthesis. According tothis method, merely the L ectoine in enantiomerically pure form isrecovered. A disadvantage of biosynthesis, however, lies in that it isrestricted to substances which are accumulated as end products in thebacteria themselves, i.e. a derivatization is at best possible inrestricted extent proceeding from the recovered ectoine.

Known in principle, too, is a chemical synthesis of ectoine according toKoichi et al. (JP-A-03031265). According to this method, ortho-trimethylacetate is reacted with 2.4-diaminobutyric acid. During the reaction,the 2.4 diaminobutyric acid is initially acetylated, and subsequently acondensing ring closure to the desired product is realized at elevatedtemperature.

Apart from ectoine and its derivates, corresponding 5- and 7-ringanalogues are known, too. The corresponding 7-ring analogue is alsodesignated as homoectoine(4,5,6,7-tetrahydro-2-methyl-1H-[1,3]-diazepine-4-carboxylic acid),calling the corresponding 5-ring analogue DHMICA(4.5-dihydro-2-methyl-imidazole-4-carboxylic acid). In conformity withthe synthesis according to Koichi, homoectoine is obtained by ringclosure between ortho-trimethyl acetate and ornithine, whereas DHMICA isobtained by ring closure between ortho-trimethyl acetate and 2.3-diaminopropionic acid. In the course of initial investigations, homoectoine andDHMICA demonstrated promising properties.

However, there still does not exist a general synthesis approach toprepare different cyclic amidines which in their basic structurecorrespond to ectoine, homoectoine or DHMICA. Hence the task posed is toprovide such a method.

This task is inventively solved by a method and process according toclaim 1.

The inventive synthesis strategy starts from a lactone of the generalformula II which is converted by bromination to a dibromine compoundIII. Bromination is accomplished in presence of bromine and PBr₃.Instead of a direct use of PBr₃ it is also possible to use phosphorous.

The 2.4-dibromine butyrate III obtained by bromination must beesterified for the subsequent reactions, thus getting to thecorresponding ester IV of the 2.4-dibromine butyrate. Esterification isimportant on the one hand in view of follow-up reactions, and moreoveresterification should be executed instantly, if possible, to prevent aformation of the corresponding α-bromine-γ-butyrolactones from thedibromine compound III under HBr-splitting-off. Esterification ispreferably realized by reaction with methanol or ethanol in an acidicenvironment. Accordingly, esterification can be performed immediatelyafter bromination, i.e. without any intermediate isolation of thecompound III, for example by adding absolute methanol or ethanol underpassage of gaseous hydrogen chloride HCl.

By a well-aimed variation of the substituent R2, R3, R4, R5 and R6 atthe butyrolactone II, correspondingly substituted ectoine derivativescan be produced. The radicals R2, R3, R4, R5 and R6 may be H oroptionally substituted alkyl, cycloalkyl, aryl, heteroaryl, alkylaryl,arylalkyl, alkoxyalkyl, alkylthioalkyl, aryloxyalkyl or arylthioalkylradicals. A broad range of possible variations is thus available.Preferably, however, the radicals R2, R3, R4, R5 and R6 are H, C₁- toC₆-alkyl or aryl. Frequently a substitution will merely exist at one ofthe items R2, R3, R4, R5 and R6, whereas the other radicals represent anH atom.

Upon preparation of the esterified dibromine compound IV, amination to adiamino compound V is realized. Substitution of bromine by amino groupscan be accomplished either by a direct reaction with ammonia or by anucleophilic substitution with an azide followed by a subsequenthydrogenation. A direct reaction with ammonia can be accomplished in aconcentrated aqueous ammonia solution at an elevated temperature ofapprox. 50° C. over an extended period of time of approx. 20 to 30hours, by, reaction with ammonia in an autoclave or in liquid ammonia.For example, in the latter case, ammonia is condensed in a flask atapprox. −40° C. and diluted with anhydrous ether, if required. Thedibromine compound IV is slowly added. The reaction occurs over a periodof approx. 2 hours.

Alternatively, a nucleophilic substitution of the bromine atoms can alsobe realized with an azide, preferably using sodium azide NaN₃.Subsequently, hydrogenation is carried out at standard conditions via anappropriate catalyst, for example palladium/activated carbon Pd/C orplatinum.

Finally, the diamino compound V thus obtained is cyclized to the desiredproduct, possibly after previous hydrolysis of the ester function. Inparticular, this can be accomplished by reaction with a suitableortho-ester R1-C(OR18)₃, with R1 again having the meaning and importanceoutlined hereinabove. R18 is an alkyl radical, more particularly aC₁-C₆-alkyl radical. The reaction is realized according to the generalscheme as had been described by Koichi et al. (see above). Ortho-esterwith R18=Me and R1=Me or Ph are obtainable on the market.

Instead of utilizing an ortho-ester, a reaction with an imidate orthioimidate is also conceivable. The reaction with a purchasable methylacetimidate (R1=Me) and/or methyl benzimidate (R1=Ph) is illustrated inthe following scheme and has been generally described by J. Einsiedel etal., Bioorganic & Medicinal Chemistry Letters, 2003, 13, 851-854.

By utilizing an appropriate ortho-ester, imidate or thioimidate, thesubstituent R1 of the desired product can be adjusted. For example, byuse of ortho-acetic acid trimethyl ester (trimethyl-orthoacetate,R1,18=methyl), an ectoine derivative in which R1=methyl is generated,too.

As has been mentioned hereinabove, a hydrolysis of the ester functioncan also be accomplished prior to or after cyclization to amidine of thegeneral formula I, i.e. R7=H upon completed hydrolysis, and it should benoted that—with a physiological pH—ectoine and its derivatives whichdispose of a carboxyl function are present as zwitterion. As a matter offact, the carboxy function can also be further derivatized by realizingan additional esterification. A broad range of possible variations isthus available to prepare different amidines of the general formula I.

Another viable derivatizing of the carboxy function lies in a conversionto amide. To this effect, in accordance with commonly known methods andprocesses, the product I, VI and/or XI is converted from a carboxylicacid and/or a carboxylic acid ester into a carboxylic acid amide. Forexample, this is accomplished by a direct conversion of ester or throughan indirect route of reacting the corresponding acid chloride and/oranhydride with ammonia/an amine. Thus, compounds of the following basicstructure are obtained:

Accordingly, R19 and R20 each can be H or alkyl, giving preference toR20=H. In particular, R19 can be a long-chain alkyl chain, for exampleC₈-, C₉-, C₁₀-, C₁₁-, C₁₂-, C₁₃-, C₁₄-, C₁₅-, C₁₆-, C₁₇- or C₁₈. At thesame time, R2-R6, R8-R14 and/or R15-R17 preferably is H, while R1 isequal to H or methyl, i.e. the rings mainly carry no furthersubstituents. Amidines of this nature appear to be particularlypromising due to the heightened lipophilicity.

The applied γ-butyrolactones are partly purchasable, for exampleR2=methyl, R3, R4, R5, R6=H (CAS: 108-29-2) or R2=phenyl, R3, R4, R5,R6=H (CAS: 1008-76-0), R2, R3, R4, R5=H, R6=methyl (CAS: 1679-47-6).Other lactones are not purchasable, but can be produced in accordancewith the methods and processes described in the relevant literature. Forexample, WO 94/12487 A1 discloses the preparation ofα-aryl-γ-butyrolactones. To this effect, an anion of the malonate as perthe formula

is reacted with an ethylene compound of the formula Y—CH₂—CH₂—OZ, with Yrepresenting a leaving group such as tosylate or mesylate, and with Zrepresenting a protective group, obtaining the compound

which under hydrolysis results in the desired lactone.

Other methods and processes which also start from malonates aredescribed in B. Hoefgen et al., J. med. Chem., 2006, 49, 760-769 or B.M. Nilsson et al., J. med. Chem. 1992, 35, 285-294. Butyrolactones witha substituent R6≠H can be illustrated, for example, in accordance withthe following scheme:

The preparation of butyrolactones with R5≠H is feasible, for example, inconformity with the following scheme:

Further examples may be gathered from S. Schulz, Liebigs Ann. Chem.1992, 8, 829-834.

In analogous manner, not only cyclical amidines with a 6-ring, but alsothose with a 5-ring or 7-ring can be produced, whose basic structurecorresponds with that of the DHMICA and homoectoine, respectively. Thereaction sequence corresponds with the sequence described hereinabove,though the basis to start from is not a γ-butyrolactone, but a β- and/orδ-lactone. This is turn is brominated and subsequently esterified. Thebromine atoms are substituted by amino groups, and the ring closure isimplemented subsequently by reaction with an ortho-ester, imidate, orthioimidate. The reaction conditions are by and large correspondingconditions, though the yield with the ring closure to a 7-ringempirically is worse, which is mainly attributable to the greater ringtension. In this manner, one obtains the 5-ring XI and/or 7-Ring VI. Thereaction schemes are illustrated in the following:

Synthesis of the homoectoine derivative VI

Synthesis of the DHMICA derivative XI:

In conformity with a particularly advantageous variant of the inventivemethod, the cyclic amidines are recovered in an enantiomerically pureform. Hence, the compounds are the following ones:

In particular, it can be the L-enantiomer so that for example thesynthetically produced ectoine corresponds with the natural L-ectoine.

To prepare a cyclic amidine in enantiomerically pure form, the diaminocompound V, X and/or XV is recovered in enantiomerically pure form andsubsequently reacted as described above with an ortho-ester, imidate orthioimidate to obtain a cyclic amidine according to the formula I, VI orXI. The centre of chirality existing in the diamino compound ismaintained.

Initially, the diamino compound is acylated at both amino functions, andsubsequently a stereoselective monodeacylation is accomplished by theaid of an aminoacylase. Merely one enantionmer of the diacylated diaminocompound is deacylated at one amino function, whereas the otherenantiomer remains acylated at both amino functions. Enyzmes suitablefor this purpose, particularly those which merely hydrolyze theL-enantiomer of an amino acid are widely known. Of the two acylatedamino groups, only the amino group in α-position is deacylated to thecarboxyl group. After the reaction with the aminoacylase, it results amixture composed of the monoacyl compound of the one enantionmer and thediacyl compound of the other enantiomer.

Subsequently, the diacyl compound of the non-desired enantiomer must besplit-off. The remaining monacyl compound of the desired enantiomer isthen hydrolyzed to the free diamino compound V, X and/or XV. Now, thereis only one enantiomer of the diamino compound present, which in thefollowing is designated as V_(enant), X_(enant) and/or. XV_(enant). Thefurther conversion to the desired, enantiomerically pure compoundI_(enant), VI_(enant) oder XI_(enant) is accomplished at describedhereinabove. All embodiments for the preparation of cyclic amidinestherefore in principle apply both to the use of the diamino compound V,X or XV as racemate or as enantiomer. Inasmuch as there is the talk ofusing an enantiomer, the optical purity may also be under 100%, thoughit is important that an enantiomer is substantially enriched as comparedwith the other enantiomer.

The acylation of the diamino compound V, X or XV in particular is anacetylation. Acylation can be carried out with usual acylating agents,for example by the aid of a carboxylic acid anhydride, a carboxylic acidchloride or a carboxylic acid bromide. Likewise, imidayolides orcarboxylic acid thiolester as well as 2-pyridine thiolester can beutilized. Moreover, there are other methods and processes knownaccording to prior art of technology in order to activate the acyl groupof a carboxylic acid in such a manner that a reaction with an aminofunction to amide can be realized, for example the conversion withdicyclohexylcarbodiimide (DCC), 2-chlorine pyridinium or3-chloroxazolium ions etc. Special preference in the case of acetylationis given to the application of acetic anhydride. This is realized in theusual manner in the alkaline range.

A selective mono-deacylation is accomplished by application of an aminoacylase. These enzymes which belong to the family of hydrolases arecapable of splitting-off the N-acyl gropu at the amino function of anamino acid, i.e. selectively with one enantiomer only. In most cases,the splitting-off is realized only with the L-enantiomer of the aminoacid which is the enantiomer that mainly occurs in nature, whereas theD-enantiomer is not affected. Therefore, such amino acylases are alsodesignated as N-acyl-L-amino acid amidohydrolase The use of acylase I,in particular of acylase I from aspergillus, is given specialpreference. This acylase is commercially obtainable, for example fromthe company Fluka, and it is also launched on the market in immobilizedform on Eupergit. In addition, by means of the acylase I fromaspergillus, only the acyl group at the amino function in α-position tothe carboxyl group is split-off, whereas, for example, in case of theN^(α),N^(γ)-diacetyl-diaminobutyric acid, the acyl group at the γ-aminofunction is not affected.

However, there are D-amino acylases known, too, which selectivelysplit-off only the N-acyl group with the D-enantiomer of an amino acid(vide e.g. C. S. Hsu et al., Protein Sci., 2002, 11, 2545-2550). Withthe use of such an acylase, the L-enantiomer remains accordinglydiacylated and can be split-off. In this manner, the inventive processprovides a viable way to corresponding D-enantiomers of the cyclicamidines to be synthesized, particularly towards the D-ectoine,D-homoectoine, and D-DHMICA.

A separation of the diacylated non-desired enantiomer of the diaminocompound is can be accomplished via a cation exchanger. To this effect,the solution is acidified with the amino acylase and the cationexchanger is washed with water. Subsequently, the desired enantiomer canbe eluted in the alkaline range, for example with an NH₃-solution.

In order to subsequently liberate also the second amino function (in γ-,δ- or β-position) from the acyl group, a conventional method of theamide hydrolysis is employed, particularly by application of an acid ora base. Preference is given to splitting-off the acyl group in theacidic environment, for example by addition of HCl. The entire route torecover an enantiomer of a diamino compound is illustrated in thefollowing by way of a 2.4 diaminobutyric acid. This acid is purchasable,but can also be obtained by applying the inventive method, with it beingrequired to execute a hydrolysis of the ester function after preparationof the diamino compound.

Apart from the inventive method, the invention also relates to cyclicamidines of the general formulae I, VI and XI as well as to saltsderived hereof, except for the natural ectoine, homoectoine, and DHMICAitself as well as some other cyclic amidines which have already beendescribed in the relevant literature. It is to be expected that thecorresponding cyclic amidines will find applicability in medical,cosmetic or biological fields similarly to the applicability of ectoineor hydroxyectoine. This includes an application as active ingredient inskin care and as sun protection, the stabilization of cells, proteins,nucleic acids and biomembranes. To be named furthermore are theprotection of cells, particular of skin cells, from stress factors suchas heat, cold, UV radiation and dryness. By the aid of the inventivemethod, however, almost any differently functionalized ectoine,homoectoine and DHMICA derivatives can be produced, whose properties canbe adapted depending on demand. For example, it is possible to integratelonger alkyl chains into the target structures I, VI and XI in order toincrease the lipophilicity of the product. This in turn can improve itsapplicability in cosmetics. Though ectoine itself is excellently solubein water which can be explained merely by its function as a compatiblesolute, in which ectoine gets enriched in high concentration in cytosol,but solubility in more richly lipophilic environments is restricted.Owing to the fact that the inventive method opens-up a broad range ofpossibilities for functionalization, the solubility of the ectoinederivative can be adjusted depending on demand.

Preferentially, the radicals R1 to R17 are H, C₁- to C₆-alkyl or aryl.In most cases, however, at least one substituent of the substituentcouples R2-R3, R4-R5, R8-R9, R10-R11, R12-R13 and R15-R16 is=H.Frequently, a substitution will exist merely at one of the positions R1to R17, whereas the other relevant radicals constitute an H atom.

Furthermore, special preference is given to such cyclic amidines of thegeneral structure I, VI and XI, in which R7 is a long-chain alkylradical, i.e. R7 preferably is a C₈-, C₉-, C₁₀-, C₁₁-, C₁₂-, C₁₃-, C₁₄-,C₁₅-, C₁₆-, C₁₇- or C₁₈-alkyl radical. At the same time, R2-R6, R8-R14and/or R15-R17 preferably is H, whereas R1 is=H or methyl, i.e. therings mainly carry no other substituents. On account of the increasedlipophilicity, such amidines appear to be particularly promising.

In other preferred embodiments, the radical R1 constitutes a short-chainalkyl radical, particularly methyl, ethyl, n-propyl, isopropyl, n-butyl,sec-butyl, isobutyl or tert-butyl. At the same time, the radicals R2-R6,R8-R14 and/or R15-R17 as well as R7 preferably are again H, subject tothe proviso that in this case R1 must not be methyl.

In accordance with another preferred embodiment, only one of theradicals R2-R5, R8-R13 and/or R15 or R16 each is=methyl, ethyl orphenyl, whereas the other radicals as well as R6, R14 and/or R17 are=H.At the same time, the following is preferentially valid for R1 and R7:R1 and R7=H; R1 and R7=methyl; R1=H and R7=methyl or R1=methyl and R7=H.

The invention furthermore also relates to cyclic amidines inenantiomerically pure form in which they can be produced according tothe modified method described hereinabove. Thus, the cyclic amidine ofthe general formula I, VI or XI is preferably present as L-enantiomer,though it is also conceivable to exist as D-enantiomer which in terms ofstereochemistry deviates from natural ectoine. Enantiomerics relates tothe centre of chirality in α-position towards the carboxylic group.Accordingly, the L-enantiomer is the enantiomer which in terms of itsstereochemistry corresponds to the natural ectoine((S)-2-methyl-1,4,5,6-tetrahydro-4-pyrimidine carboxylic acid), with itbeing necessary to take into account that with a neutral pH valueectoine is present as zwitterion.

The following examples serve as a further explanation of the presentinvention:

EXAMPLE 1 Synthesis of methyl-2-4-dibrombutyrate (2,4-dibromine butyricacid methyl ester

25 ml bromine were slowly added in droplets to a reheated mixture ofphosphorous tribromide (0.83 ml) and γ-butyrolactone (42 g, 0.49 mol) ata temperature of 100 to 115° C. Subsequently, the mixture was reheatedfor another period of 30 min. The mixture was cooled in an icy bath,carefully adding 83 ml methanol and introducing gaseous HCl. Thesolution thus obtained was reheated for a period of 48 hours to 50° C.The volatile constituents were withdrawn, and the remaining oil wasdiluted with diethyl ester and washed two times with a 3%-rich aqueoussodium hydrogen carbonate solution and an NaCl solution. The washingsolutions were re-extracted with diethyl ether and the unified organicphases were concentrated in a vacuum. Distillation at 0.2 torr resultedin the product methyl-2.4-dibromine butyrate in form of a colourlessliquid (100.4 g, 80%).

EXAMPLE 2 Synthesis of 2.4-diamino butyric acid methyl ester

At a temperature of −40° C., 150 ml ammonia were condensed into a flaskand diluted with 150 ml anhydrous diethyl ether. Slowly added to thissolution were 100 g of 2.4-dibromine butyric acid methyl ester (0.385mol) in 100 ml anhydrous diethyl ether. At a temperature of −40° C.stirring was continued for 2 hours, and subsequently it was slowlyreheated to ambient temperature. After stirring over night, the ammoniumbromide was filtered-off and the solvent was withdrawn. Obtained were43.3 g (85%) 2.4-diamino butyric acid methyl ester.

EXAMPLE 3 Synthesis of1,4,5,6-tetrahydro-2-methyl-pyrimidine-4-carboxylic acid methyl ester

A solution of 2.4 g (0.018 mol) 2.4-diamino butyric acid methyl esterand 3.77 g ortho-acetic acid trimethyl ester in 50 ml dried methanolwere reheated in the reflux for 24 hours. The solution was concentratedto dryness and the product was recrystallized from methanol/ethylacetate. Obtained were 1.12 g (7.2 mmol, 40%)1,4,5,6-tetrahydro-2-methyl-pyrimidine-4-carboxylic acid methyl ester.

EXAMPLE 4 Synthesis of L-2.4-diamino butyric acid

1) N^(α),N^(γ)-diacetyl-D,L-diamino butyric acid

Under stirring and ice cooling, acetic acid anhydride (1.42 ml; 15 mmol)and 7.5 ml 2 N NaOH were added in 5 portions to a solution ofD,L-2.4-diamino butyric acid (955 mg; 5 mmol) in 7.5 ml 2 N NaOH. Byadding 1.5 ml 2 N NaOH, the pH value was adjusted to 7.5. After 1 hourin ice, the solution was acidified with 2 ml 37% HCl to pH 3 and theaqueous phase was extracted 2 times with 50 ml n-butanol/ethyl acetate(2:1) each. After evaporating the organic phase in a vacuum to a smallvolume, precipitation was effected with methyl-tert-butylether Obtainedwere 800 mg (79%) N^(α),N^(γ)-diacetyl-D,L-diamino butyric acid as acolourless solid. DC: R_(f)=0.6 in acetonitrile/water/acetic acid(30:10:5); ESI-MS: m/z=203.05 [M+H]⁺; M_(r)=202.21 calcld. for C₈H₁₄N₂O₄

2) N^(γ)-acetyl-L-diamino butyric acid

N^(α),N^(γ)-diacetyl-D,L-diamino butyric acid (404 mg; 2 mmol) wereadulterated in 20 ml Sörensen-phosphate-buffer (1/15 molar) at pH 7.6with 28 mg acylase I aspergillus (Fluka, 0.72 U7 mg enzyme; thiscorresponds to 10 U/1 mmol substrate) and incubated for 20 hours in awater bath at 40° C. The enzyme was separated by means of a membranefilter Amicon Ultra-4 (exclusion limit 10 kD) and the solution wasadjusted with 1 N HCl to pH 2.5.

After feeding it to 50 ml cation exchanger Dowex 50Wx8 (50-100 mesh),washing was carried out with water having a pH value of up to 5(separation of the diacetyl compound) and subsequently the desiredproduct was eluted with 1 N ammonia. The eluate was evaporated in avacuum, wherby colourless crystals of the N^(γ)-acetyl-L-diamino butyricacid developed instantly. Yield: 140 mg (88% of the expected 50%-richyield); DC: R_(f)=0.3 mm acetonitrile/water/acetic acid (30:10:5);ESI-MS: m/z=161.10 [M+H]⁺; M_(r)=160.17 calcld. for C₆H₁₂N₂O₃

3) L-diamino butyric acid

N^(γ)-acetyl-L-diamino butyric acid (140 mg; 0.87 mmol) were stirred in10 ml 2 N HCl under reflux. Upon evaporation in vacuum, the crystallineresidue was dissolved in as little water as possible and precipitatedwith 4 ml hot ethanol abs. The L-diamino butyric acid was obtained asmonohydrochloride colourless. Yield: 125 mg (93%); DC: R_(f)=0.1 inacetonitrile/water/acetic acid (30:10:5); ESI-MS: m/z=119.08 [M+H]⁺;M_(r)=118.14 calcld. for C₄H₁₀N₂O₂; M_(r)=154.60 calcld. forC₄H₁₀N₂O₂×HCl; [α]²⁰ _(D)=+24.4° (c=1.2 in 6 N HCl); Lit. (Beilstein,4/IV, 2613): [α]²¹ _(D)=+23.8° (c=1 in 6 N HCl); Fluka catalogue: [α]²⁰_(D)=24±2° (c=1 in 6 N HCl). The enantiomer purity of the amino acid wasconfirmed and verified by means of derivatization with Marlfey's reagent(J. G. Adamson et al., Anal. Biochem., 1992, 202, 210-214) and asubsequent HPLC. Conditions: Column ET 125/4 Nucleosil 100-5 C8(Macherey and Nagel, Düren); linear gradient from eluent A (1%trifluoroacetic acid (TFA) in H₂0) to eluent B (0.8% TFA inacetonitrile); R_(t)=15.6 min.

1. Method for preparation of cyclic amidines of the general formula I

with R1, R2, R3, R4, R5, R6, R7=H, optionally substituted alkyl,cycloalkyl, aryl, heteroaryl, alkylaryl, arylalkyl, alkoxyalkyl,alkylthioalkyl, aryloxyalkyl or arylthioalkyl comprised of the followingsteps: Bromination of a lactone of the general formula II

to a dibromine compound of the general formula III

Esterification of the dibromine compound III to a compound IV

Amination of the dibromine compound IV to a diamino compound V

and Reaction of the compound V with an ortho-ester of the generalformula R1-C(OR18)₃, with R1 having the meaning and importance outlinedhereinabove and R18=alkyl, particularly C₁-C₆-alkyl, or an imidate ofthe general formula

With R1, R18 having the meaning and importance as outlined hereinabove,or a thioimidate of the general formula

with R1, R18 having the meaning and importance as outlined hereinabove,to obtain a cyclic amidine of the general formula I.
 2. Method forpreparation of cyclic amidines of the general formula VI

with R1, R7, R8, R9, R10, R11, R12, R13, R14=H, optionally substitutedalkyl, cycloalkyl, aryl, heteroaryl, alkylaryl, arylalkyl, alkoxyalkyl,alkylthioalkyl, aryloxyalkyl or arylthioalkyl comprised of the followingsteps: Bromination of a lactone of the general formula VII

to a dibromine compound of the general formula VIII

Esterification of the dibromine compound VIII to a compound IX

Amination of the dibromine compound IX to a diamino compound X

and Reaction of the compound X with an ortho-ester of the generalformula R1-C(OR18)₃, with R1 having the meaning and importance outlinedhereinabove and R18=alkyl, particularly C₁-C₆-alkyl, or an imidate ofthe general formula

With R1, R18 having the meaning and importance as outlined hereinabove,or a thioimidate of the general formula

with R1, R18 having the meaning and importance as outlined hereinabove,to obtain a cyclic amidine of the general formula I.
 3. Method forpreparation of cyclic amidines of the general formula XI

with R1, R7, R15, R16, R17=H, optionally substituted alkyl, cycloalkyl,aryl, heteroaryl, alkylaryl, arylalkyl, alkoxyalkyl, alkylthioalkyl,aryloxyalkyl or arylthioalkyl comprised of the following steps:Bromination of a lactone of the general formula XII

to a dibromine compound of the general formula XIII

Esterification of the dibromine compound XIII to a compound XIV

Amination of the dibromine compound XIV to a diamino compound XV

and Reaction of the compound XV with an ortho-ester of the generalformula R1-C(OR18)₃, with R1 having the meaning and importance outlinedhereinabove and R18=alkyl, particularly C₁-C₆-alkyl, or an imidate ofthe general formula

with R1, R18 having the meaning and importance as outlined hereinabove,or a thioimidate of the general formula

with R1, R18 having the meaning and importance as outlined hereinabove,to obtain a cyclic amidine of the general formula XI.
 4. Methodaccording to claim 1, characterized in that the ester function of thecompounds V, X or XV is hydrolyzed prior to the step of the reactionwith orthoester, imidate or thioimidate.
 5. Method according to claim 1,characterized in that the amination of the dibromine compound IV, IX orXIV is realized by reaction with an azide and a subsequenthydrogenation.
 6. Method according to claim 1, characterized in that theamination of the dibromine compound IV, IX or XIV is realized byreaction with NH₃.
 7. Method according to claim 1, characterized in thatR1-17 is=H, C₁- to C₆-alkyl or aryl.
 8. Method according to claim 1,characterized in that at least one substituent of the substituentcouples R2-R3, R4-R5, R8-R9, R10-R11, R12-R13 and R15-R16 is=H. 9.Method according to claim 1, characterized in that the carboxylic acidor carboxylic acid ester function COOR7 is converted to a carboxylicacid amide function CONR19R20, with R19 and R20 being=H or alkyl. 10.Method according to claim 1, characterized by the following additionalsteps to recover an enantiomer of the compounds V, X or XV: Acylation ofthe compounds V, X or XV at both amino functions to a diacyl compoundStereoselective monodeacylation of an enantiomer of the diacyl compoundin α-position by application of an amino acylase to a monoacylcompound-Separation of the non-deacylated diacyl compound, andHydrolysis of the monoacyl compound to the enantiomer of the compoundsV, X or XV.
 11. Method according to claim 10, characterized in that theenantiomer is the L-enantiomer.
 12. Method according to claim 10,characterized in that the acyl group is an acetyl group.
 13. Methodaccording to claim 10, characterized in that the amino acylase is anacylase I from aspergillus.
 14. Cyclic amidine of the formula I as wellas its salts

with R1, R2, R3, R4, R5, R6, R7=H, optionally substituted alkyl,cycloalkyl, aryl, heteroaryl, alkylaryl, arylalkyl, alkoxyalkyl,alkylthioalkyl, aryloxyalkyl or arylthioalkyl subject to the provisothat R1 is not=methyl, if R2, R3, R4, R5, R6 are=H and R7 is=H, methylor ethyl, R1 is not=phenyl, if R2, R3, R4, R5, R6, R7 are=H, R1 is not2′-pyridyl, if R2, R3, R4, R5, R6, R7 are=H and R1 is not n-propyl, ifR2, R3, R4, R5, R6, R7 are=H.
 15. Cyclic amidine of the formula VI aswell as its salts

with R1, R7, R8, R9, R10, R11, R12, R13, R14=H, optionally substitutedalkyl, cycloalkyl, aryl, heteroaryl, alkylaryl, arylalkyl, alkoxyalkyl,alkylthioalkyl, aryloxyalkyl or arylthioalkyl, subject to the provisothat R1 is not=methyl, if R7, R8, R9, R10, R11, R12, R13, R14 are=H. 16.Cyclic amidine of the formula XI as well as its salts

with R1, R7, R15, R16, R17=H, optionally substituted alkyl, cycloalkyl,aryl, heteroaryl, alkylaryl, arylalkyl, alkoxyalkyl, alkylthioalkyl,aryloxyalkyl or arylthioalkyl, subject to the proviso that R1 isnot=methyl, if R7, R15, R16, R17 are=H.
 17. Cyclic amidine according toclaim 14, characterized in that R7 is a C₈-, C₉-, C₁₀-, C₁₁-, C₁₂-,C₁₃-, C₁₄-, C₁₅-, C₁₆-, C₁₇- or C₁₈-alkyl radical and that R1 is eitherH or methyl, with R2, R3, R4, R5 and R6 in case of a 6-ring amidine, R8,R9, R10, R11, R12, R13 and R14 in case of a 7-ring amidine, and R15,R16, and R17 in case of a 5-ring amidine being=H.
 18. Cyclic amidineaccording to claim 14, characterized in that R1 is=ethyl, n-propyl,isopropyl, n-butyl, sec-butyl, isobutyl or tert-butyl and R7 is=H, withR2, R3, R4, R5 and R6 in case of a 6-ring-amidine, R8, R9, R10, R11,R12, R13 and R14 in case of a 7-ring amidine and R15, R16 and R17 incase of a 5-ring amidine being=H.
 19. Cyclic amidine according to claim14, characterized in that in case of the 6-ring amidine only one of theradicals R2, R3, R4 or R5 is=methyl, ethyl or phenyl, whereas the otherradicals as well as R6 are=H, in case of the 7-ring amidine only one ofthe radicals R8, R9, R10, R11, R12 or R13 are=methyl, ethyl or phenyl,whereas the other radicals as well as R14 are=H, in case of the 5-ringamidine only one of the radicals R15 or R16 is=methyl, ethyl or phenyl,whereas the other radical as well as R17 are=H, with the following beingvalid for R1 and R7: R1 and R7=H or R1 and R7=methyl or R1=H andR7=methyl or R1=methyl and R7=H.
 20. Cyclic amidine according to claim14, characterized in that the amidine is present as L- or D-enantiomer.21. Cyclic amidine of the formulae XVI, XVII or XVIII as well as itssalts

with R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, R12, R13, R14, R15,R16, R17 being=H, optionally substituted alkyl, cycloalkyl, aryl,heteroaryl, alkylaryl, arylalkyl, alkoxyalkyl, alkylthioalkyl,aryloxyalkyl or arylthioalkyl, and with R19 and R20 being=H or alkyl.22. Cyclic amidine according to claim 21, characterized in that R19is=C₈-, C₉-, C₁₀-, C₁₁-, C₁₂-, C₁₃-, C₁₄-, C₁₅-, C₁₆-, C₁₇- or C₁₈-Alkyland R2, R3, R4, R5, R6, R8, R9, R10, R11, R12, R13, R14, R15, R16, R17and R20 being=H, with R1 being=H or methyl.
 23. Cyclic amidine accordingto any one of the formulae reflected hereinafter as well as its salts